Expanding the map of Pol III transcriptome: eCLIP and ROPE-seq of POLR3A
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE337525 | GEO | 2026/07/09
REPOSITORIES: GEO
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Project description:RNA polymerase III (Pol III) synthesizes noncoding RNAs essential for translation, RNA processing, and transcription regulation. Its activity aligns with cellular growth and nutrient availability, and its dysregulation is linked to diseases like breast cancer and leukodystrophy. RNA polymerase III composition is shaped by the mutually exclusive incorporation of two paralogous subunits, RPC7α and RPC7β, encoded by genes POLR3G and POLR3GL in vertebrates. The expression of POLR3G and POLR3GL is spatiotemporally regulated during development, and multiple reports point to RPC7α-enhanced Pol III activity patterns, indicating that Pol III identity may underlie dynamic Pol III transcription patterns observed in higher eukaryotes. In cancer, upregulation of POLR3G, but not POLR3GL, is associated with poor survival outcomes among patients, suggesting differences between RPC7α and RPC7β further influence disease progression and may translate into future biomarkers and therapeutic strategies.
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Project description:RNA polymerase III (Pol III) synthesizes noncoding RNAs essential for translation, RNA processing, and transcription regulation. Its activity aligns with cellular growth and nutrient availability, and its dysregulation is linked to diseases like breast cancer and leukodystrophy. Despite its importance, Pol III regulators remain largely unidentified due to a lack of exploration of Pol III-centered protein-protein interactions (PPIs). To address this need, we conducted high throughput proteomic experiments and identified over 300 novel Pol III interactors, including Pol II-related transcription factors, nuclear import factors, and splicing factors. Most notably, our proteomic survey uncovers complex-complex interactions between Pol III and the NuRD (Nucleosome Remodeling and Deacetylase) complex. NuRD complex is a known Pol II co-repressor, but its recruitment to and effect on Pol III remains uncharacterized. Using innovative large-scale NuRD binding pattern meta-study, NuRD ChIP-seq combined with Pol III inhibition, and Pol III activity profiling in mammalian cell lines, we revealed that the NuRD is recruited by Pol III itself and fine-tunes Pol III transcription activity. We found that NuRD exhibits a binding preference for highly active Pol III genes, and inhibition of Pol III transcription reduced the enrichment of NuRD subunits. Functionally, inhibiting NuRD deacetylase activity releases its inhibition on Pol III transcription and increases its dynamic range. In addition, our ATAC-seq analysis suggests NuRD is potentially involved in establishing nucleosome positioning through its ATP-dependent remodeler activity. Beyond identifying NuRD as a novel Pol III regulator, our survey of Pol III interactions unexpectedly identifies physical interactions between Pol III and mRNA splicing factors. We demonstrate that Pol III depletion leads to rewiring of splicing efficiencies for specific mRNA subpopulations, suggesting a previously unrecognized interplay between Pol III and RNA processing factors. Together, these findings advance our understanding of Pol III interactome, uncovering new layers of transcriptional control and unexpected functional consequences of Pol III interactions in human cells.
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Project description:RNA polymerase III (Pol III) synthesizes noncoding RNAs essential for translation, RNA processing, and transcription regulation. Its activity aligns with cellular growth and nutrient availability, and its dysregulation is linked to diseases like breast cancer and leukodystrophy. Despite its importance, Pol III regulators remain largely unidentified due to a lack of exploration of Pol III-centered protein-protein interactions (PPIs). To address this need, we conducted high throughput proteomic experiments and identified over 300 novel Pol III interactors, including Pol II-related transcription factors, nuclear import factors, and splicing factors. Most notably, our proteomic survey uncovers complex-complex interactions between Pol III and the NuRD (Nucleosome Remodeling and Deacetylase) complex. NuRD complex is a known Pol II co-repressor, but its recruitment to and effect on Pol III remains uncharacterized. Using innovative large-scale NuRD binding pattern meta-study, NuRD ChIP-seq combined with Pol III inhibition, and Pol III activity profiling in mammalian cell lines, we revealed that the NuRD is recruited by Pol III itself and fine-tunes Pol III transcription activity. We found that NuRD exhibits a binding preference for highly active Pol III genes, and inhibition of Pol III transcription reduced the enrichment of NuRD subunits. Functionally, inhibiting NuRD deacetylase activity releases its inhibition on Pol III transcription and increases its dynamic range. In addition, our ATAC-seq analysis suggests NuRD is potentially involved in establishing nucleosome positioning through its ATP-dependent remodeler activity. Beyond identifying NuRD as a novel Pol III regulator, our survey of Pol III interactions unexpectedly identifies physical interactions between Pol III and mRNA splicing factors. We demonstrate that Pol III depletion leads to rewiring of splicing efficiencies for specific mRNA subpopulations, suggesting a previously unrecognized interplay between Pol III and RNA processing factors. Together, these findings advance our understanding of Pol III interactome, uncovering new layers of transcriptional control and unexpected functional consequences of Pol III interactions in human cells.
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Project description:The mechanism of assembly of RNA polymerase III (Pol III), the 17-subunit enzyme that synthesizes tRNAs, 5S rRNA, and other small-nuclear (sn) RNAs in eukaryotes, is not clearly understood. The recent discovery of the HSP90 co-chaperone PAQosome (Particle for Arrangement of Quaternary structure) revealed a function for this machinery in the biogenesis of nuclear RNA polymerases. However, the connection between Pol III subunits and the PAQosome during the assembly process remains unexplored. Here, we report the development of a mass spectrometry-based assay that allows the characterization of Pol III assembly. This assay was used to dissect the stages of Pol III assembly, to start defining the function of the PAQosome in this process, to dissect the assembly defects driven by the leukodystrophy-causative R103H substitution in POLR3B, and to discover that riluzole, an FDA-approved drug for alleviation of ALS symptoms, partly corrects these assembly defects. Together, these results shed new light on the mechanism and regulation of human nuclear Pol III biogenesis.
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Project description:RNA polymerase III (Pol III) is an essential enzyme responsible for the synthesis of several small non-coding RNAs, a number of which are involved in mRNA translation. Recessive mutations in POLR3A, encoding the largest subunit of Pol III, cause POLR3-related hypomyelinating leukodystrophy (POLR3-HLD), characterized by deficient central nervous system myelination. Identification of the downstream effectors of pathogenic POLR3A mutations has been so far elusive. Here, we used CRISPR-Cas9 to introduce the POLR3A mutation c.2554A>G (p.M852V) into human cell lines and assessed its impact on Pol III biogenesis, nuclear import, DNA occupancy, transcription, and protein levels. Transcriptomic profiling uncovered a subset of transcripts vulnerable to Pol III hypofunction, including a global reduction in tRNA levels. The brain cytoplasmic BC200 RNA (BCYRN1), involved in translation regulation, was consistently affected in all our cellular models, including patient-derived fibroblasts. Genomic BC200 deletion in an oligodendroglial cell line led to major transcriptomic and proteomic changes, having a larger impact than those of POLR3A mutations. Upon differentiation, mRNA levels of the MBP gene, encoding myelin basic protein, were significantly decreased in POLR3A-mutant cells. Our findings provide the first evidence for impaired Pol III transcription in cellular models of POLR3-HLD and identify several candidate effectors, including BC200 RNA, having a potential role in oligodendrocyte biology and involvement in the disease.
2019-03-20 | GSE118739 | GEO
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Project description:The packaging of the genetic material into chromatin imposes the remodeling of this barrier to allow efficient transcription. RNA polymerase II activity is associated with several histone modification complexes that enforce remodeling. How RNA polymerase III (Pol III) counteracts the inhibitory effect of chromatin is unknown. We report here that antisense RNA Polymerase II (Pol II) transcription is critical to prime and maintain nucleosome depletion at Pol III loci and allow efficient Pol III recruitment upon re-initiation of growth from stationary phase. Antisense Pol II is recruited by the Pcr1 transcription factor, which affects local histone occupancy through the associated SAGA complex and a Pol II phospho-S2 CTD / Mst2 pathway. These data expand the central role of Pol II in gene expression beyond mRNA synthesis.
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Project description:MAF1 represses Pol III-mediated transcription by interfering with TFIIIB and Pol III. Herein, we found that MAF1 knockdown induced CDKN1A transcription and chromatin looping concurrently with Pol III recruitment. Simultaneous knockdown of MAF1 with Pol III or BRF1 (subunit of TFIIIB) diminished the activation and looping effect, which indicates that recruiting Pol III was required for activation of Pol II-mediated transcription and chromatin looping. ChIP analysis after MAF1 knockdown indicated enhanced binding of Pol III and BRF1, as well as of CFP1, p300, and PCAF, which are factors that mediate active histone marks, along with the binding of TBP and POLR2E to the CDKN1A promoter. Simultaneous knockdown with Pol III abolished these regulatory events. Similar results were obtained for GDF15. Our results reveal a novel mechanism by which MAF1 and Pol III regulate the activity of a protein-coding gene transcribed by Pol II.
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